Antitumor and Antibacterial Activity of Ni(II), Cu(II), Ag(I), and Hg(II) Complexes with Ligand Derived from Thiosemicarbazones: Characterization and Theoretical Studies

Four new complexes (Ni2+, Cu2+, Ag+, and Hg2+) were prepared from the ligand N-(4-chlorophenyl)-2-(phenylglycyl)hydrazine-1-carbothioamide (H2L). Analytical and spectroscopic techniques were used to clarify the structural composition of the new chelates. In addition, all chelates were tested against bacterial strains and the HepG2 cell line to determine their antiseptic and carcinogenic properties. The Ni(II) complex was preferable to the other chelates. Molecular optimization revealed that H2L had the highest reactivity, followed by Hg-chelate, Ag-chelate, Ni-chelate, and Cu-chelate. Moreover, molecular docking was investigated against two different proteins: the ribosyltransferase enzyme (code: 3GEY) and the EGFR tyrosine kinase receptor (code: 1m17).


ESI-MS Spectra
The mass spectrum of the ligand exhibited a molecular ion peak at m/z = 336 amu (Calc. m/z = 334.5  Figure S3).

ESI-MS Spectra
The mass spectrum of the ligand exhibited a molecular ion peak at m/z = 336 amu (Calc. m/z = 334.5  Figure S3).
The interpretation of the mass spectra of the complexes Cu(II) and Ag(I) has been clarified, revealing several significant fragments. The data are in a good agreement with the proposed molecular formulas for complexes Cu(II) and Ag(I) (calc. 732.18 and 618.65, respectively), and this verifies the complexes' chemical structures.
The mass spectra of the Cu(II) complex shows the peak attributed to the molecular ion peak [M+] m/z at 732 (8.52%) corresponding to C 30

Electronic Spectral Bands
The electronic spectral bands of the H 2 L and Ni 2+ , Cu 2+ , Ag + , and Hg 2+ complexes (λmax, nm) in the DMF solution were scanned in the range of 190-810 nm at room temperature. The values of the λmax and magnetic moments (µeff) are recorded in Table 3. The UV spectra of ligand were observed as two absorption bands at 260 and 300 nm assigned to the π-π* transition [3]. On complexations, the UV-visible spectra of Ni(II), Cu(II), Ag(I), and Hg(II) complexes were shifted to higher wavelength exhibit bands at 286 and 374, 281 and 371, 281, and 299 and 378 nm, respectively, which may be assigned to π -π*, n-π* transitions, and showed no d-d band, representing a square planar geometry for Cu 2+ , Ag + , and Hg 2+ complexes, although Ag compounds were known mostly as two-coordinated, but the square planar silver(I) complexes increasingly observed stereochemistry for silver(I), as was found in our synthesized complex [14][15][16]. In contrast, the Ni(II) complex is square pyramidal geometry. The difference in the λ max values of the H 2 L and its complexes can approve the coordination of the ligand with metal ions [17,18].

PXRD of Ligand and Metal Complexes
The X-ray diffract gram patterns of ligand H 2 L and its complexes of Ag(I) and Hg(II) complexes were evaluated in Table 4 and Figures 2, 3 and S2. The powder diffraction patterns were recorded over the (2θ = 5-90) range lattice constants. The intensities of the powder lines and the corresponding 2θ values are found to be different between ligand and complexes, indicating their crystalline nature. The average particle size of the crystalline ligand (H 2 L) and its complexes was calculated using Scherer's equation [19][20][21]. The Scherer's constant (K) in the formula provides for the particle's shape and is commonly assumed to be 0.9. It was found that the calculated crystalline size was in the nano range. The values of the crystallite size for H 2 L, 3S, and 4S are 41.50, 35.12, and 41.02, 54.35, 52.03, and 26.75, and 52.28, 50.11, and 64.12 nm, respectively.

Thermal Analysis
TGA plays a crucial role in assessing the features of new metal complexes, discovering the different solvent molecules inside or outside the coordination sphere, and figuring

Thermal Analysis
TGA plays a crucial role in assessing the features of new metal complexes, discovering the different solvent molecules inside or outside the coordination sphere, and figuring

Thermal Analysis
TGA plays a crucial role in assessing the features of new metal complexes, discovering the different solvent molecules inside or outside the coordination sphere, and figuring out the thermal stability of the complexes. In addition to the microanalyses' outcomes, the TGA results, which were carried out between 20 and 800 • C, were utilized to assess and compute mass loss. TGA analyses were carried out for ligand and Ni 2+ (1S), Cu 2+ (2S), Ag + (3S), and  (Table 5). At temperatures between 190 and 633 • C (Calc. 100 %, found 99.9 %), the ligand TGA plot shows their complete thermal breakdown in one step. Three weight loss events were visible in the TG curves of the Ni(II) complex. The first decomposition step occurred between 41 and 178 • C and was accompanied by weight losses of 3.82 (3.86): Calc./ found %, which are interpreted as losses of the hydrated water molecule. The second phase occured between 178 and 288 • C and involved the losses of the C 15 H 14 N 4 OS and moiety with weight losses from the complexes estimated to be 63.27 (63.21) Calc./ found %. In the third phase, the temperature was between 288 and 391 • C with weight losses of 17.06 (17.11) Calc./found % corresponding to HCl and CO 2 molecules losses, and NiO remaining as the final residue. While in the Cu(II), Ag(I), and Hg(II) complexes, the TGA plot showed the complete decomposition of organic molecules in one step between a temperature range of 105 and 385 • C with weight losses of 91.32, 56.72, and 59.65 (91.28, 56.76, 59.60) Calc./ found % and the remainder of Cu, 2AgO + 3C, and HgO + 6C as the final residues, respectively. Table 6 revealed the geometric information of the H 2 L and its metal chelates as the energy, dipole moment, hardness, softness, chemical potential, and electronegativity. Researchers were investigating the frontier molecular orbitals LUMO (p acceptor) and HOMO (p donor) that represents the charge transfer interface inside the LUMO-HOMO molecule leading to the appearance of the parameters, the hardness and softness, which are commonly used as a criterion of chemical reactivity and stability ( Figure 4). The smaller hardness values imply a higher reactivity, which means that a molecule with a small HOMO-LUMO gap is more reactive and softer. The hardness and softness can be calculated using the equations η(hardness) = (I − A)/2; S(softness) = 1/2η. The compounds' reactivity is arranged as follows: H 2 L > Hg-chelate > Ag-chelate > Ni-chelate > Cu-chelate. The energy gap for all the complexes is higher than that of the ligand. So, the investigated complexes are more stable than the parent ligand; accordingly, the Cu-chelate was the most stable. The geometrical molecular structure provided the atomic ordering of H 2 L, and complexes are also included ( Figure 5). After investigating the computed bond lengths and orientations of ligand and its metal chelates, we observed some changes after coordination, as presented in Table S1. Furthermore, many bond lengths were elongated as N12-N11, C9-O10, C8-N7, and C13-N12 to adjust for the coordination via the N12 and O10 in all chelates. The thione group shared in the coordination in both 1S and 3S chelates with the formation of new M-S bonds.

Biological Applications
The antibacterial activities of ligand H2L and Ni 2+ , Cu 2+ , Ag + , and Hg 2+ complexes were screened against bacterial species, Gram-negative bacteria (Escherichia coli and Klebsiella pneumonia), and Gram-positive bacteria (Staphylococcus aureus and Streptococcus mutants). Ampicillin and gentamicin were used as the standards for antibacterial studies. The results of the antibacterial activity of the ligand and their complexes are presented in Table S2 and Figure 6. These results suggested that the complexes are more potent antibacterial agents than ligands due to their chelation ability. Whether ampicillin and In the case of Ni, Ag, and Hg chelates, every metal completed its coordination with other chelating agents and the parent ligand, such as carbonate, water, and iodide and hydroxide. Table 6 illustrates the bond lengths of newly constructed ligand bonds. Additionally, new bond angles were observed, and others were changed to optimize the coordination as C16-N15-C13, N15-C13-Nl2, C13-N12-Nl1, and O10-C9-N11. The negative charge is delocalized over N12 and O10 with calculated charges of −0.473 and −0.459, respectively. Therefore, both are common donation sites in all chelates. After chelation, these charges were decreased, and the electron density above the metal ions increased due to the charge transfer from ligand to metal. The charges of N12 and O10 were changed to −0.395 and −0.345, −0.458 and −0.479, −0.391 and −0.300, and −0.465 and −0.362 in 1S, 2S, 3S, and 4S, respectively. It was noticed that some electron densities were increased due to the back donation from M to the ligand. The metal charges changed to +0.336, +0.456, +0.129, and +0.519 for 1S, 2S, 3S, and 4S, respectively.

Antibacterial Activity
The antibacterial activities of ligand H 2 L and Ni 2+ , Cu 2+ , Ag + , and Hg 2+ complexes were screened against bacterial species, Gram-negative bacteria (Escherichia coli and Klebsiella pneumonia), and Gram-positive bacteria (Staphylococcus aureus and Streptococcus mutants). Ampicillin and gentamicin were used as the standards for antibacterial studies. The results of the antibacterial activity of the ligand and their complexes are presented in Table  S2 and Figure 6. These results suggested that the complexes are more potent antibacterial agents than ligands due to their chelation ability. Whether ampicillin and gentamicin were used as standard drugs, the nickel complex in our study had the best antibacterial activity against bacterial species. The nickel complex showed more antimicrobial effects against Escherichia coli and Klebsiella pneumonia than gentamicin. This agrees with other researchers using nickel in their studies [21,22]. Similarly, it showed more antimicrobial effects against Staphylococcus aureus and Streptococcus mutans than ampicillin. This is in line with other similar research results [23]. Moreover, the mercury complex has a high antimicrobial effect against Staphylococcus aureus and Streptococcus mutans with an inhibition zone of 23.3 ± 0.6 and 44.3 ± 0.6, respectively, and is higher than that of the reference antibiotic. Furthermore, ampicillin has inhibition zones of 22 ± 0.1 and 30 ± 0.5 when testing Staphylococcus aureus and Streptococcus mutans, respectively. This is consistent with other studies that have used mercury [24,25]. Consequently, the resulting data showed that the antibacterial efficacy of new complexes against the Gram-negative bacteria could be arranged in the following order: Ni complex > gentamicin > Hg complex > Ag complex > Cu complex > Ligand. However, for Staphylococcus aureus, the sequence of antibacterial action was Ni complex > Hg complex > ampicillin > Cu complex > Ag complex > ligand. The same pattern was observed against Streptococcus mutans, except that the ligand has no antimicrobial activity. The effectiveness of synthesized compounds may appear to be lipophilic, which may block or impede the viable development of Gram-negative and Gram-positive bacteria by facilitating the diffusion of the compounds through the lipid bilayer membrane. This is explained by the chelation theory, which argues that a decrease in the polarizability of metal complexes or an increase in hydrogen bonding can increase the lipophilic susceptibility of the complex, hence enhancing its antimicrobial activity [26]. and has no antimicrobial activity. The effectiveness of synthesized compounds may appear to be lipophilic, which may block or impede the viable development of Gram-negative and Gram-positive bacteria by facilitating the diffusion of the compounds through the lipid bilayer membrane. This is explained by the chelation theory, which argues that a decrease in the polarizability of metal complexes or an increase in hydrogen bonding can increase the lipophilic susceptibility of the complex, hence enhancing its antimicrobial activity [26]. Figure 6. In vitro antimicrobial activity of (H2L) and its metal complexes against Gram-negative and Gram-positive bacteria.

Cytotoxicity
The MTT test was used to assess the in vitro cytotoxicity of the ligand H2L and its complexes with Ni(II), Cu(II), Ag(I), and Hg(II) against the human HepG2 cell line. In terms of optical thickness, the movement of mitochondrial dehydrogenase was determined to be an indication of cell viability. Non-linear regression methods were used to determine the IC50 values for the investigated substances during the experiment. The results are reported as the IC50, which is the concentration of a chemotherapeutic agent that produces a 50% reduction in cancer cell proliferation compared to control cell growth [27].

Cytotoxicity
The MTT test was used to assess the in vitro cytotoxicity of the ligand H 2 L and its complexes with Ni(II), Cu(II), Ag(I), and Hg(II) against the human HepG2 cell line. In terms of optical thickness, the movement of mitochondrial dehydrogenase was determined to be an indication of cell viability. Non-linear regression methods were used to determine the IC 50 values for the investigated substances during the experiment. The results are reported as the IC 50 , which is the concentration of a chemotherapeutic agent that produces a 50% reduction in cancer cell proliferation compared to control cell growth [27]. The cytotoxicity for the ligand was performed at concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 µg/mL. Moreover, the findings showed that the ligand and its complexes are potent against human HepG2 cell lines. Importantly, the Ni(II) complex was the most effective and exhibited concentration-dependent effects, suggesting its potential utility in cancer treatment.

Molecular Docking Studies
Simulation of the protein-drug interaction is important in designing the structurebased drug [28,29]. Thus, we investigated the theoretical interaction between the prepared compounds with some proteins selected from the protein data bank. The ribosyltransferase enzyme (code: 3GEY) was selected for the antibacterial study, and the EGFR tyrosine kinase receptor (code: 1m17) was selected for the anticancer study. The interaction profile

Molecular Docking Studies
Simulation of the protein-drug interaction is important in designing the structurebased drug [28,29]. Thus, we investigated the theoretical interaction between the prepared compounds with some proteins selected from the protein data bank. The ribosyltransferase enzyme (code: 3GEY) was selected for the antibacterial study, and the EGFR tyrosine kinase receptor (code: 1m17) was selected for the anticancer study. The interaction profile of the tested compounds with 3GEY and 1m17 is presented in Tables S4 and S5 and Figures 8 and 9. The antibacterial docking results revealed the potency of the examined compounds, as indicated by the negative values of the scoring energies or the different types of interactions, as presented in Figure 8. There are several interactions, most notably the side chain acceptor in all chelates and the arene cation interaction type, prominent in numerous compounds such as H 2 L, 1S, and 2S. Table S4 reflects the interactions for each compound.  H 2 L ligand: the backbone donor interaction between Leu-A624 and the oxygen amide group of the ligand. Additionally, arene cation is an interaction between Lys-A518 and the phenyl group of the parent ligand. Ni-chelate: backbone donor interaction between Asn-A553 and the carbonyl oxygen of the carbonate group, sidechain acceptor between Gln-A521 and the H atom of the NH group, and arene-arene between Phe-A642 and phenyl ring. Cu-chelate: arene-cation interaction between Lys-A525 and the phenyl ring, sidechain acceptor between Asp-A623 and Asn-B508, the H atom of the NH group, and a sidechain donor between the Gln-B549 oxygen amide group. Ag-chelate: arene-cation interaction between Lys-A518 and two of the phenyl rings and sidechain acceptor between Asn-B508 and (H) of the water molecule. Hg-chelate: sidechain acceptor between Asn-B508 and (H) of the NH group. Despite the fact that Cu-chelate has the highest scoring energy, experimentally, Ni-chelate had the highest antibacterial activity. It may be due to the various interactions observed only in Ni-chelate as the sharing of the carbonate group with a backbone donor interaction and the aromatic-aromatic interaction via the phenyl ring.
The investigation of the EGFR tyrosine kinase receptor (code: 1m17) explored the high affinity of the prepared compounds to the 1m17 protein. Hg-chelate had the highest significant negative scoring energy value that is compatible with the IC 50 of chelates. The different types of interactions are represented in Figure 9 and Table S5. The chelates show side chain donor (arene-cation and sidechain acceptor) and metal contact interactions for Ni-, Cu-, and Hg-chelates. H2L ligand: the backbone donor interaction between Leu-A624 and the oxygen am ide group of the ligand. Additionally, arene cation is an interaction between Lys-A518 an the phenyl group of the parent ligand. Ni-chelate: backbone donor interaction betwe Asn-A553 and the carbonyl oxygen of the carbonate group, sidechain acceptor betwe Gln-A521 and the H atom of the NH group, and arene-arene between Phe-A642 and ph nyl ring. Cu-chelate: arene-cation interaction between Lys-A525 and the phenyl rin sidechain acceptor between Asp-A623 and Asn-B508, the H atom of the NH group, and sidechain donor between the Gln-B549 oxygen amide group. Ag-chelate: arene-cation i teraction between Lys-A518 and two of the phenyl rings and sidechain acceptor betwe Asn-B508 and (H) of the water molecule. Hg-chelate: sidechain acceptor between As B508 and (H) of the NH group. Despite the fact that Cu-chelate has the highest scorin energy, experimentally, Ni-chelate had the highest antibacterial activity. It may be due the various interactions observed only in Ni-chelate as the sharing of the carbonate grou with a backbone donor interaction and the aromatic-aromatic interaction via the phen ring. Finally, all bond lengths for most of the interactions were found to be less than 3.5 Å, the reported range for an actual docking track [30]. The obtained data indicated that the examined compounds are promising and recommend further bioactivity investigations.

Material and Methods
The preparation tools and methods utilized for the structure confirmation and application of metal salts NiCO 3 , Cu(ClO 4 ) 2 , Ag 2 S, and HgI 2 were obtained as shown in the Supplementary Material and in Section S1.

Preparation of Ligand and Metal Complexes
The organic ligand N-(4-chlorophenyl)-2-(phenylglycyl)hydrazine-1-carbothioamide was synthesized and characterized according to Aly and Eldourghamy [21] and Abdalla et al. [3]. Ni 2+ , Cu 2+ , Ag + , and Hg 2+ chelates were prepared by adding a stoichiometrically amount of target metal ions in absolute ethanol to a hot ligand solution in a 1:1 molar ratio as represented in Scheme 1. The metal complexes were magnetically stirred at 60 • C for 5-9 h. The hot precipitous were filtered, leaving the solution at 35 • C to vaporize some solvents and promote the crystallization. The crystals were collected by vacuum filtrations, washed several times with anhydrous diethyl ether, and dried under a vacuum in the presence of phosphorus pentoxide (P 4 O 10 ).

Computational Study
GaussView 5.0.8 (Wallingford, CT, USA, 2009) software was used to prepare th put files of compounds, as shown in Section 2 [31].

Antibacterial Assay
The antimicrobial activity of the prepared ligands and complexes(1S-4S) was ducted using the agar well diffusion method [32,33]. The H2L and complexes (1S-4S) confirmed in vitro for their antibacterial activity against staphylococcus a (ATCC:13565) and Streptococcus mutans (ATCC:25175) (Gram-positive bacteria), E richia coli (ATCC:10536) and klebsiella pneumonia (ATCC:10031) (Gram-negative bact All strains were stored at −80 °C before culture preparation. The strains were cultur a suitable culture media; Staphylococcus aureus and Streptococcus mutans were cultur blood agar, while Escherichia coli and klebsiella pneumonia were cultured in McConkey Details of the antimicrobial screening methodology are illustrated in the suppleme file (Section S3) [34].

Computational Study
GaussView 5.0.8 (Wallingford, CT, USA, 2009) software was used to prepare the input files of compounds, as shown in Section 2 [31].

Antibacterial Assay
The antimicrobial activity of the prepared ligands and complexes(1S-4S) was conducted using the agar well diffusion method [32,33]. The H 2 L and complexes (1S-4S) were confirmed in vitro for their antibacterial activity against staphylococcus aureus (ATCC:13565) and Streptococcus mutans (ATCC:25175) (Gram-positive bacteria), Escherichia coli (ATCC:10536) and klebsiella pneumonia (ATCC:10031) (Gram-negative bacteria). All strains were stored at −80 • C before culture preparation. The strains were cultured in a suitable culture media; Staphylococcus aureus and Streptococcus mutans were cultured in blood agar, while Escherichia coli and klebsiella pneumonia were cultured in McConkey agar. Details of the antimicrobial screening methodology are illustrated in the Supplementary Material (Section S3) [34].

Cytotoxicity Assays
The MTT assay [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is a colorimetric examination for measuring the cell metabolic activity and proliferation [35]. In the current research, the MTT assay was used to test the cytotoxicity of the prepared ligand and its chelates against the HepG2 cell line. More details of the antitumor screening methodology are illustrated in the Supplementary Material (Section S4) [36][37][38].

Conclusions
The characterization and biological activity of four new (Ni 2+ , Cu 2+ , Ag + and Hg 2+ ) prepared complexes were studied. The outcomes revealed the following: All complexes have a square planar geometry, while Ni(II) is square pyramidal. Depending on the HOMO-LUMO energy gap, the DFT analysis generated that they were more stable complexes than the parent ligand.
Ni complexes are the most potent antimicrobial agent against different bacterial strains of Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, and Streptococcus mutans with inhibition zones of 65.95 ± 0.5, 57.36 ± 0.6, 69.21 ± 0.6, and 72.34 ± 0.5, respectively, which are higher than the compared reference antibiotics gentamicin (for Gram-negative) and ampicillin (for Gram-positive). Moreover, the ligand and its complexes (1S-4S) exhibit substantial anticancer effects.
Molecular docking explored the suggested interactions with the active amino acids of the ribosyltransferase and EGFR tyrosine kinase enzymes.